The present invention describes peptide substrates of the metalloproteases ADAM8, ADAM15 and MDC-L. The invention also describes methods using these peptides to discover pharmaceutical agents that modulate these proteases. The invention further describes CD23 processing activity for these enzymes that may have important therapeutic implications for the use of inhibitors of these enzymes in allergic diseases such as asthma.
The disintegrin metalloprotease (or ADAM) family of cell surface proteolytic enzymes is known to play roles in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, modulation of Notch receptor and ligand processing, and processing of the pro-inflammatory cytokine, TNFxcex1. The TNFxcex1-converting enzyme, TACE or ADAM17, is currently a target for anti-inflammatory drugs (McGeehan et al., 1997; Sekut and Connolly, 1998), and other members of this family are likely to be good therapeutic targets. The genes for ADAM8, ADAM15 and MDC-L have been cloned and shown to contain the consensus sequence for an active metalloprotease, but their in vivo substrates are unknown. No proteolytic activity has been demonstrated for these proteins, apart from one reference to unpublished observations that murine ADAM8 expressed in yeast cells showed MMP-3-like, substance P degrading activity (K. Matsuura, unpublished, reported in (Yamamoto et al., 1999).
ADAM8 has been reported to be expressed almost exclusively in cells of the immune system, particularly B-cells, monocytes and granulocytes. Furthermore, its expression has been shown to be inducible by LPS and xcex3-interferon (Kataoka et al., 1997). ADAM8 is specifically expressed in eosinophils, one of the most important effector cell types at the site of inflammation in allergic asthma. ADAM8 therefore represents a therapeutic target for human diseases, such as for the treatment of allergy and/or asthma.
ADAM15 is a membrane-bound disintegrin metalloprotease containing an RGD integrin-binding sequence, which may function in cell adhesion through binding to integrin xcex1vxcex23 (Nath et al., 1999; Zhang et al., 1998), and two proline-rich sequences, shown to interact with SH3 domains in endophilin-I and SH3PX1 (Howard et al., 1999). ADAM15 is found in human aortic smooth muscle and cultured umbilical vein endothelial cells. While ADAM15 is not expressed in normal blood vessels, it has been detected in developing atherosclerotic lesions (Herren et al., 1997), and has also been shown to be upregulated in osteoarthritic versus normal human cartilage (Bohm et al., 1999). Thus ADAM15 plays a role in the atherosclerosis and/or cartilage degeneration. ASAM15 therefore represents a therapeutic target for human diseases, such as for the treatment of osteoarthritis and atherosclerosis.
A member of the ADAM family, MDC-L, was recently cloned and shown to be specifically expressed by lymphocytes in two alternative forms, a membrane-bound form, MDC-Lm, and a secreted protein, MDC-Ls (Roberts et al., 1999). The lymphocyte- specific expression of MDC-L suggests that it may have an important immunological function, but its in vivo substrate(s) are unknown and proteolytic activity has not been previously demonstrated.
Excessive production of IgE is believed to be a major mediator of allergic responses(Corry and Kheradmand, 1999), resulting in pathophysiology ranging from inflammation to severe bronchoconstriction. IgE binds to two different receptors, the high affinity IgE receptor found on mast cells and basophils, and CD23, the low affinity IgE receptor, expressed on the surface of B cells (Corominas et al., 1993), monocytes(Alderson et al., 1992), macrophages (Melewicz et al., 1982), and eosinophils(Sano et al., 1999). The latter receptor, CD23, is subject to proteolytic release of soluble extracellular fragments (Letellier et al., 1989), which have been shown to cause upregulation of IgE production (Aubry et al., 1992; Yu et al., 1994)and induction of inflammatory cytokines(Armant et al., 1994; Armant et al., 1995). Increased levels of soluble CD23 have been observed in allergic asthma (Di Lorenzo et al., 1999; Monteseirin et al., ; Yanagihara et al., ), in chronic B-lymphocytic leukemia (Beguin et al., ; Dine et al., ; Knauf et al., 1997) and in rheumatoid arthritis(Bansal et al., 1994; Chomarat et al., 1993; Ribbens et al., 2000).
The implication of soluble CD23 in the upregulation of IgE production suggests that inhibition of the enzyme(s) responsible for CD23 processing may offer a therapeutic approach for the treatment of allergic diseases. In support of this theory, it has been shown that inhibition of CD23 proteolysis can significantly inhibit IgE release in B cell lines (Christie et al., 1997; Wheeler et al., 1998), and in mouse models of IgE production and bronchoconstriction in response to ovalbumin challenge (Christie et al., 1997; Dasic et al., 1999).
The production of soluble CD23 is mediated by a membrane-bound metalloprotease (Marolewski et al., 1998), which can be inhibited by compounds that do not affect matrix metalloprotease activity(Bailey et al., 1998; Bailey et al., 1999). The prior art does not identify the specific metalloprotease that cleaves CD23 to generate the upregulation of allergic response, thus this remains an unmet need in the development of therapeutics to treat human immune diseases such as allergy.
The present invention describes peptide substrates of the metalloproteases ADAM8, ADAM15 and MDC-L, represented by the peptides in Table 1. The peptides of the present invention are useful in assays to identify modulators of the activity of ADAM8, ADAM15 and MDC-L, particularly those compounds that inhibit ADAM8, ADAM15 and MDC-L enzymatic activity.
The present invention also demonstrates that ADAM8, which is found on the same cell types as CD23, is able to process membrane-bound CD23 on human macrophage and B cell lines. Thus the present invention provides methods to measure the decrease of cell-surface CD23 or increase of soluble fragments of CD23 produced by the enzymatic activity of ADAM8, ADAM15, or MDC-L. Further the present invention provides assays to test putative modulators of ADAM8, ADAM15, or MDC-L enzymatic activity towards CD23.